Comments (3)
Hi,
thank you so much for the help! It works great now :)
Best!
Dimitri
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The amplicon sequence is the following: GCAGGGGGCAAAGAGAGGCCGGGGAGCAAGGAGGAGGTGGATGAAGACCGCGACGTCGATGAGTCCTCCCCCCAAGACTCCCCTCCCTCCAAGGCCTCCCCAGCCC
The gRNA is the following: GGGAGGACTCATCGACGTCG
And the fastq sample example is attached.
APOE34-MAPT1-CR-clone35-C11-DB-FR-NIM_S124_L001_R1_001.fastq.gz
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Hi @dbudinger
Sorry about the confusion. I took a look at the fastq file you sent. The first read does indeed contain the amplicon, but it also contains a lot of other sequence before and after the amplicon sequence:
read: GCTTCTAGGAGACCTGCACNAGGAGGGGCCGCCGCTGAAGGGGGCAGGGGGCAAAGAGAGGCCGGGGAGCAAGGAGGAGGTGGCTGAAGACCGCGACGTCGATGAGAGCACCCCCCAAAACTCCCCTCCCTCCAAGACGTCCCCAGCCCACGATGGGAGGCCTCCCCAGACAGCCGCCAGAGAATCCACCAGCATACTGTCTCTTATACACATCTCCGAGCCCACGAGACGATGCCTTACATCTCGTATGCCGTCTTCTGCTTGAAAAAAAAAAAAAAAAACACAACGGACGACCACAGAC
amplicon: GCAGGGGGCAAAGAGAGGCCGGGGAGCAAGGAGGAGGTGGATGAAGACCGCGACGTCGATGAGTCCTCCCCCCAAGACTCCCCTCCCTCCAAGGCCTCCCCAGCCC
CRISPResso requires reads to align to the amplicon with a minimum 60% similarity, and because this alignment only has 106bp/301bp = 35% similarity, it is discarded.
In order to run CRISPResso, you need to trim the reads to only contain the amplicon sequence.
You can use this doing cutadapt. For example, I created a trimmed file with the following commands:
cutadapt -g GCTTCTAGGAGACCTGCACNAGGAGGGGCCGCCGCTGAAGGGG APOE34-MAPT1-CR-clone35-C11-DB-FR-NIM_S124_L001_R1_001.fastq.gz -o half.fastq.gz -e 0.2
cutadapt -a ATGGGAGGCCTCCCCAGACAGCCGCCAGAGAATCCACCAGCATACTGTCTCTTATACACATCTCCGAGCCCACGAGACGATGCCTTACATCTCGTA -e 0.2 half.fastq.gz -o trimmed.fastq.gz
This runs successfully and produces the following output.
Let me know if you'd like me to trim other files for you.
from crispresso2.
Related Issues (20)
- Alleles frequency table plot not generated HOT 3
- INDELs not recognized in the allele frequency table HOT 2
- CRIPSResso2 package not included in channel HOT 1
- How to solve Error 0? HOT 4
- Allele plot doesn't show base letter HOT 2
- Confused about sequence directions in prime editing parameters HOT 6
- ERROR: Offset around cut would be greater than reference sequence length. HOT 2
- CRISPRessoAgregate
- ERROR: NUMPY RELATED HOT 1
- A small mistake in 'CRISPResso_quantification_of_editing_frequency.txt' HOT 1
- Question about CRISPResso for WGS - and potential feature HOT 1
- High % of "AMBIGUOUS" reads – what are they? HOT 3
- Numpy Error HOT 6
- ERROR:HDR number in the plot not the same as the number in txt file HOT 2
- @N-yating If you pin to Python 2.7, it will install an old version of CRISPResso2. I would recommend using Python 3 for the most recent version of CRISPResso2.
- CRISPResso2 breaks because on lack of pin in matplotlib version HOT 3
- http://crispresso2.pinellolab.org/ - 502 Bad gateway HOT 2
- CRISPRessoAggregate failure HOT 2
- amplicon for Targeted Sequencing
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